Various neurodegenerative and developmental disorders of unknown etiology characteristically affect specific neural systems. These diseases have been hypothesized to involve defects in trophic interactions that are essential for maintaining the connectivity of a particular system. In order to study the role of trophic influences in the development and maintenance of cholinergic innervation of cortex, and to provide insight into the selective vulnerability of cholinergic neuronal networks in neurologic disease, the experiments outlined in this proposal will employ clonal cell lines and rotation-mediated culture methods to study the trophic interactions between cholinergic neurons of the septal region of the basal forebrain and their target cells in the hippocampus. In preliminary studies, cell lines expression specific cholinergic and neuronal characteristics, including choline acetyltransferase and neurofilament expression, neurite formation, and the ability to aggregate in rotation-mediated culture, have been derived from septal cells using somatic cell fusion techniques. Recently, clonal cell lines have also been derived from hippocampal target areas. In preparation for studies employing these lines in conjunction with the rotation-mediated culture system, the target-specific influences of primary hippocampal cells on primary septal cholinergic cells in that culture system will be further characterized. Studies will be performed to extend the initial observation that mouse embryonic hippocampal cells, in contrast to nontarget cerebellar cells, enhance septal cholinergic cell survival and fiber proliferation in reaggregate cultures, and promote the formation of synaptic contacts by septal cholinergic cells with identified target cell populations. The hybrid cell lines will be further characterize with respect to the expression of additional cholinergic markers. The capacity to respond to target-specific influences on fiber proliferation and synapse formation and maintenance will be assessed, as will responsiveness to nerve growth factor (NGF). Permanent cell lines have also been derived from hippocampal target areas. These will be characterized and screened for the expression of either NGF- or non-NGF-mediated trophic effects on both primary septal cholinergic neurons and cholinergic cell lines. If any of the hippocampal cell lines exhibit facilitory effects that are not attributable to NGF, the relevant macromolecule(s) will localized to a particular subcellular fraction. The active fraction will be employed to prepare specific antibodies, and through iterative immunization techniques, the relevant macromolecule(s) will be isolated and partially characterized. These studies will provide a clearer understanding of the factors involved in the formation and maintenance of specific connections between basal forebrain cholinergic neurons and their cortical targets, and important insights into the possible causes of neuronal loss and dysfunction in disorders affecting this central cholinergic system.